Method of screening for substance preventing or treating diseases in association with malfunction of IL-6 family cytokine receptor malfunction

ABSTRACT

The invention provides a method of screening for substances preventing or treating diseases in association with IL-6 family cytokine receptor malfunction, which comprises using a transgenic mouse or a part thereof expressing a gp130 variant, wherein said gp130 variant comprises a substitution or a deletion of a tyrosine residue, which corresponds to the tyrosine residue at position 759 of human gp130 protein, or a substitution, an insertion or a deletion of one or more amino acid residues in a region comprising said tyrosine residue.

This application is a U.S. national stage of International ApplicationNo. PCT/JP01/06464 filed Jul. 27, 2001.

TECHNICAL FIELD

The invention relates to a method of screening for substances preventingor treating diseases in association with malfunction of IL-6 familycytokine receptors, which comprise using of a transgenic mouse or a partthereof.

BACKGROUND ART

Rheumatoid arthritis (RA) is a chronic inflammatory disease accompaniedwith polyarthritis as a main lesion and a high prevalence ofcollagenosis (1% of the global population). Cause of the disease isunknown, but the disease is believed to be the result of environmentalfactors, such as infection, in addition to genetic factors. Correlationbetween development of symptom and HLA DR4 and the existence of T cellsin synovial tissue suggest that abnormalities in T cells participate inthe pathophysiology. Moreover, abnormalities in immunity, such ashyperglobulinemia due to polyclonal activation of B cells and appearanceof autoantibodies, are observed. To date, model mice for autoimmunedisease accompanied with arthritis and arthritis-induced model mice havebeen employed to analyze the pathophysiology of the disease, andtransgenic mice and spontaneous mice which present arthritis have beenreported in recent years. For example, the following are typicalarthritis model mice reported to date.

1. Arthritis Model Mouse Induced by Antigenic Sensitization Etc.

The arthritis is induced by immunization of an intra-articularautoantigen with a strong adjuvant, or injection of microbial antigencrossreactive to the intra-articular autoantigen. Unlike RA, thesymptoms are observed in a short period of time after the sensitization.The microbial antigen used is epidemiologically unrelated to human RA.

(1) Type II Collagen (IIC)-Induced Arthritis Model Mouse (Nature,666–668, 1980. J. S. Courtenay et. al.)

Procedures: DBA/1J mouse is immunized with an emulsion prepared frombovine IIC and Freund's complete adjuvant. Three weeks later, bovine IICand Freund's incomplete adjuvant are boosted, and the arthritis developsin 1–2 weeks.

Features: Unlike the adjuvant arthritis in rat, which is similar toReiter's syndrome in human, this model is considered to be more relevantto human RA, in that it has no cutaneomucosa and organ involvement, andobservations of the tissues resemble closely those in human RA. Cellularimmunity and humoral immunity participate in this model mouse, andtherefore, the arthritis can be induced with an anti-collagen monoclonalantibody. IL-6 is a precipitating factor (J. Exp. Med. 187, 461–468,1998, Arthritis Rheumat. 42, 1635–1999). This model mouse is useful inproducing various knock-out mice to identify gene products thataggravate or relieve arthritis.

Problems: Non-physiological operations for immunization are required.Deterioration-improvement pattern as observed in human RA is notobserved. Rheumatoid factor (RF) is not produced.

(2) Antigen-Induced Arthritis Mouse (Arthritis Rheum. 20, 841–850, 1977)

Procedures: The arthritis is induced by sensitizing a mouse with anantigen, such as methylated bovine serum albumin, and followed byboosting of the antigen intra-articularly.

Features: IL-6 is a precipitating factor (Proc. Natl. Acad. Sci. USA,95, 8222–8226, 1998). C57BL/6 and Balb/c mouse are used.

(3) CpG-Induced Arthritis Mouse (Nature Medicine, 5, 702–705, 1999,Arthritis Rheum. 43, 356–364, 2000)

Procedures: The arthritis is induced by intra-articular injection ofCpG, which is a bacterial DNA having immuno-stimulating action.

Features: Macrophage precipitates in the arthritis. Local production ofTNFα, IL-12, IL-1β, MCP-1, and RANTES in a joint, and increasedproduction of blood IgG and IL-6 are involved. The arthritis can beinduced in C57BL/6, Balb/c, C3H/HeN mouse, etc.

2. Spontaneous Arthritis Model Mouse

(1) MRL-lpr/lpr and gld Mouse

Features: Abnormal proliferation of T cells, SLE-like lesion, arthritisand production of RF observed in this model are similar to thoseobserved in human RA. It has been clearly shown that the mainabnormalities are caused by Fas/Fas ligand.

Problems: Influences of a hereditary background involve symptoms that donot develop in C57BL/6, C3H, and AKR etc., and thus, development of thearthritis is not only due to abnormality in FAS/FAS ligand. Prolongedobservation and analysis of progression of the disease are difficult,since the animal dies at about six-months old, because of abnormalmultiplication of T cells.

(2) SKG Mouse (Reference: Molecular Medicine, vol 34, Extra Number,Immunity 1997–1998, p214–221)

Features: Symptoms of the arthritis are marked in comparison withMRL-lpr/lpr mouse. No abnormal proliferation of T cells is involved.Hyperglobulinemia is presented, and autoantibodies such as anti-IICantibody and RF of IgM class are produced. The abnormalities in T cellsare considered as the main cause because arthritis develops in a normalmouse by transplantation of T cells from a mouse presenting arthritis.This SKG mouse is derived from Balb/c mouse.

Problems: The cause of development of the disease is unknown.

3. Genetic-Engineering Model Mouse

Since the arthritis develops as a result of genetic engineering (genetransfer, knock-out, knock-in), the cause of development of the diseaseis clear.

(1) TNFα Transgenic Mouse (EMBO J. 10, 4025, 1991)

Features: This is a model mouse wherein the arthritis is caused byoverproduction of cytokines. It has been shown that the arthritis isrelieved with the anti-TNF antibody. In the hereditary background ofDBA/1, the arthritis is more serious (J. Immunol. 159, 2867–2876, 1997).IL-1β and IL-6 are produced by cells in synovial membrane. The cells insynovium tissue are mainly neutrophils, and other fibroblasts andendothelial cells, and there is little participation of lymphocytes.

(2) HTLV-1Tax Transgenic Mouse (Science, 253, 1026–1028, 1991, J.Immunol, 155, 1588–1598, 1995, J. Immunol, 161, 6592–6598, 1998)

Features: Production of cytokines such as TNF, IL-1, and IL-6 areincreased, because of the over-expression of Human T Cell Leukemia VirusType 1 gene tax. Hyperglobulinemia is presented, and RF, anti-IIC andanti-DNA antibodies are produced. Development of symptoms is not relatedto H-2 haplotype. The incident is high in order of Balb/c, C3H/HeN, andC57BL/6.

(3) IL-1 Receptor Antagonist Knock-Out Mouse: (J. Exp. Med. 191,313–320, 2000)

Features: The arthritis is developed in the hereditary background ofBalb/c. The incident is low in C57BL/6. The arthritis develops, becauseof the deficiency of a molecule which negatively controls a citokinesignal. Local production of IL-6 and IL-1β increases in a joint.Hyperglobulinemia is presented, and RF, anti-IIC and anti-DNA antibodiesare produced.

(4) T Cell Antigen Receptor Transgenic Mouse (Cell, 87, 811–822, 1996,Immunity 10, 451–461, 1999, Science, 286, 1732–1735, 1999)

Features: A transgenic mouse, which has a T cell receptor (α, β) thatI-Ak-restrictively recognizes bovine pancreas ribonuclease peptideR41-61, is crossed with a NOD mouse to develop arthritis. Neutrophilicinfiltration is observed in a joint space. Hyperglobulinemia andanti-DNA antibodies are observed, but RF is not observed. The arthritisis possible to be developed in a healthy mouse with introduction of theantibody in the serum of a mouse presenting arthritis, and it has beenshown clearly that the antigen, which is recognized by this antibody andT cells of the mouse presenting arthritis, is such antigen as referredto as Glucose-6-Phosphate Isomerase (GPI). It is associated withmechanisms wherein ribonuclease-specific T cells recognize GPI combinedwith I-Ag7 inherent in NOD mouse and are activated to induce ainflammatory response specific to a join.

The model mice described above present the symptoms of arthritis by themechanisms that are respectively different, such that they reflect thatthe pathogenesis of human RA, which is caused by many factors. Althoughall pathophysiology shown in these model mouse are not completelyconsistent with those in human RA, it is apparent that a new arthritismodel mouse would be useful in analysis of a certain aspect of the onsetof RA, in view of the fact that multiple factors participate indevelopment of RA.

Participation of IL-6 in the pathophysiology of arthritis has been shownnot only by clinical observations, but also in the model mice describedabove. However, there is no case wherein the arthritis is actuallydeveloped in any IL-6 related genetic mouse reported to date. Moreover,although it has been produced by many model mice presenting arthritis,which have been gene-engineered to enhance the production of cytokinesand their function, there are few among these model mice that aresuitable for analysis of the functional abnormalities of T cells. The Tcell antigen receptor transgenic mouse described above could notdemonstrate how self-reactivity T cell clones are selected from the Tcell repertories to break the self-tolerance. Although abnormalitiessuch as T cell selection may participate in SKG mouse, it is difficultto investigate the cause of the disease because of spontaneousdevelopment of the symptoms.

gp130 is a membrane protein having a molecular weight of 130 kDa and acommon receptor subunit for L-6 family citokines. gp130 participates innot only IL-6 signal transmission but also signal transmissions of otherIL-6 family citokines: leukemia inhibitory factor (LIF), ciliaryneurotrophic factor (CNTF), oncostatin M (OSM), Interleukin-11 (IL-11),and cardiotrophin-1 (CT-1). (Hirano, T. et al. (1997) Cytokine GrowthFactor Rev. 8, 241–52).

IL-6 family cytokine binds to a receptor, and thereby homodimerformation between gp130 proteins or heterodimer formation between gp130protein and another gp130 related factor is induced. Subsequently, atyrosine residue on the gp130 protein is phosphorylated, and SHP2 (SH2domain contained protein tyrosine phosphoatase 2) is associated with thephosphorylated tyrosine residue, and the SHP2 is further phosphorylatedto transmit signals downstream. The phosphorylation of SHP2 necessitatesphosphorylation of tyrosine 759 in human gp130 (Hirano, T. et al. (1997)Cytokine Growth Factor Rev. 8, 241–52). Furthermore, it was reportedrecently that the molecule called SOCS-3 (suppressor of cytokinesignaling-3), which is responsible for feedback control of the cytokinesignal, binds to this tyrosine 759 of human gp130 protein (Proc. Natl.Acad. Sci. USA, 2000, 97, 6493–8).

The amino acid sequence of human gp130 protein is known, and cDNAencoding the human gp130 protein has been reported (Hibi, M et al.,(1990) Cell 63, 1149–57). Also, the amino acid sequence of mouse gp130is known, and cDNA encoding the mouse gp130 has been reported (Saito etal., (1992) J. Immunol. 148, 4066–71).

DISCLOSURE OF INVENTION

The present invention provides a screening method utilizing a newpathologic model for substances preventing or treating diseases inassociation with malfunction of IL-6 family cytokine receptor.

The present invention is based on the discovery that a transgenic mouse,wherein SHP2-mediated gp130-dependent signaling cascade is selectivelydisrupted, exhibits symptoms characteristic in autoimmune disease,arthritis and rheumatoid arthritis as the result of malfunction of IL-6family cytokines.

Thus, the present invention provides

1) A method of screening for substances preventing or treating diseasesin association with IL-6 family cytokine receptor malfunction, whichcomprises using a transgenic mouse or a part thereof expressing a gp130variant, wherein said gp130 variant comprises a substitution or adeletion of a tyrosine residue, which corresponds to the tyrosineresidue at position 759 of human gp130 protein, or a substitution, aninsertion or a deletion of one or more amino acid residues in a regioncomprising said tyrosine residue;

2) A method of screening for substances preventing or treatingautoimmune diseases, which comprises using a transgenic mouse or a partthereof expressing a gp130 variant, wherein said gp130 variant comprisesa substitution or a deletion of a tyrosine residue, which corresponds tothe tyrosine residue at position 759 of human gp130 protein, or asubstitution, an insertion or a deletion of one or more amino acidresidues in a region comprising said tyrosine residue;

3) A method of screening for substances preventing or treatingarthritis, which comprises using a transgenic mouse or a part thereofexpressing a gp130 variant, wherein said gp130 variant comprises asubstitution or a deletion of a tyrosine residue, which corresponds tothe tyrosine residue at position 759 of human gp130 protein, or asubstitution, an insertion or a deletion of one or more amino acidresidues in a region comprising said tyrosine residue;

4) A method of screening for substances preventing or treatingrheumatoid arthritis, which comprises using a transgenic mouse or a partthereof expressing a gp130 variant, wherein said gp130 variant comprisesa substitution or a deletion of a tyrosine residue, which corresponds tothe tyrosine residue at position 759 of human gp130 protein, or asubstitution, an insertion or a deletion of one or more amino acidresidues in a region comprising said tyrosine residue;

5) A method of screening for antiinflammatory drugs, which comprisesusing a transgenic mouse or a part thereof expressing a gp130 variant,wherein said gp130 variant comprises a substitution or a deletion of atyrosine, which corresponds to the tyrosine residue at position 759 ofhuman gp130 protein, or a substitution, an insertion or a deletion ofone or more amino acid residues in a region comprising said tyrosineresidue;

6) The method of screening according to any one of claims 1–5, whereinthe SHP2-mediated gp130-dependent signaling cascade is selectivelydisrupted in said transgenic mouse or a part thereof expressing a gp130variant;

7) The method of screening according to any one of claims 1–5, whereinthe negative regulation by SOCS-3 of the gp130-dependent signalingcascade is disrupted in said transgenic mouse or a part thereofexpressing a gp130 variant.

8) The method of screening according to any one of claims 1–7, whereinsaid gp130 variant comprises a substitution or a deletion of a tyrosineresidue, which corresponds to the tyrosine residue at position 759 ofhuman gp130 protein.

9) The method of screening according to claim 8, wherein saidsubstitution of a tyrosine residue, which corresponds to the tyrosineresidue at position 759 of human gp130 protein, is the result of a pointmutation of the gp130 gene; and

10) The method of screening according to claim 9, wherein phenylalanineis encoded in place of tyrosine as the result of said point mutation.

The disease which develops in the transgenic mouse of the inventionappears to be caused by not only selective disruption of theSHP2-mediated gp130-dependent signaling cascade, but also malfunction offeedback control of the cytokine signalling by binding of SOCS-3.Moreover, any other unknown signaling molecule requiring tyrosine 759 ofhuman gp130 (or any tyrosine residue corresponding thereto) mightparticipate in development of the disease in the transgenic mouse of theinvention. The diseases which develop in the transgenic mouse of theinvention appear to be caused by one or more of these unknown signalingmolecules which do not work.

Considering that there is no case wherein arthritis has actuallypresented, the inventors' knowledge that arthritis is caused by a pointmutation in a gene for gp130, which is a common receptor subunit forIL-6 family cytokines, which provides valuable information. Moreover,based on the inventors' findings that a cytokine receptor simultaneouslytransmits not only multiple signals, but also sometimes signals opposingeach other, the transgenic mouse of the invention would be a uniquemodel mouse demonstrating that the disease can be caused by onlydisruption of the balance of these multiple signals.

Furthermore, it would be likely that some of the actual cases of RArelated diseases had been caused in the similar manner as in thetransgenic mouse of the invention, considering that the transgenic mousecould present arthritis with only a point mutation. Moreover,considering that arthritis could be caused by transplantation of spleencells of the transgenic mouse of the invention into alymphocyte-deficient RAG2 knock-out mouse, and that living/proliferationof the activated CD4+ T cells is observed in this RAG2 knock-out mouse,the arthritis developed in the transgenic mouse of the invention islikely caused by abnormalities in immunity principally involved with Tcells. Indeed, this is suggested from the fact that T cells expressingboth CD4 and CD8 (i.e., CD4/CD8 double positive cell) decrease and Tcells expressing CD4 alone (i.e., CD4 single positive cell) increase inthe thymus of the transgenic mouse of the invention, and that CD4+ Tcells expressing activated antigens on their surfaces increase in thethymus and peripheral lymphocytes.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows antibody levels of the anti-ssDNA antibody and theanti-dsDNA antibody in mice at 12 months of age. Open circle:gp130^(WT/WT) mouse (n=6), triangle: gp130^(WT/F759) mouse (n=5), closedcircle: gp130^(WT/WT) mouse (n=11). Horizontal bars indicate the meanvalues.

FIG. 2 shows incidences of arthritis in mice.

FIG. 3 is macroscopic observation of arthritis in gp130^(F759/F759)mouse (a copy of ankle photograph of a 9.5-month-old mouse). Left: ankleof control mouse (gp130^(WT/WT) mouse). Right: ankle ofgp130^(F759/F759) mouse.

FIG. 4 shows pathological pictures of an ankle of gp130^(F759/F759)mouse (15-month-old: hematoxylin-eosin stain of synovial tissue (×40)).Left: control mouse (gp130^(WT/WT) mouse). Right: gp130^(F759/F759)mouse. The arrowhead indicates fibroblasts growth, and the arrowindicates neutrophilic infiltration.

FIG. 5 shows pathological pictures of a knee joint of gp130^(F759/F759)mouse (15-month-old: hematoxylin-eosin stain of synovial tissue (×100)).Left: control mouse (gp130^(WT/WT) mouse). Right: gp130^(F759/F759)mouse. JS: joint space. The arrowhead indicates neutrophilicinfiltration accompanied with fibrin deposition, and the arrow indicatesthickening of synovial tissue accompanied with neutrophilic infiltrationand fibroblasts growth.

FIG. 6 shows pathological pictures of a liver of gp130^(F759/F759) mouse(hematoxylin-eosin stain of synovial tissue (×100)). Left upper: liverof control mouse (gp130^(WT/F759) mouse) (9.5-month-old). Right upper:liver of gp130^(F759/F759) mouse (9.5-month-old). Right middle: liver ofgp130^(F759/F759) mouse (15.5-month-old). The arrow indicatesdegeneration of hepatocytes. Right lower: lymph node ofgp130^(F759/F759) mouse (15.5-month-old).

FIG. 7 shows the results of flow cytometry analysis of a thymus ofgp130^(F759/F759) mouse. Thymus cells of a mouse were multiply stainedwith a fluorescence-labeled monoclonal antibodies, and the fluorescenceintensities were measured by flow cytometer. Vertical and lateral axesindicate the fluorescence intensities reflecting the binding of thefluorescence-labeled antibodies against the surface antigens, which areindicated in the vertical and lateral direction on the left side of thepanels, respectively, and thus, the expression levels of the surfaceantigens are indicated for the individual cells. Upper panels: theresults of analysis of CD4 and CD8 expressions. Lower panels: theresults of analysis of IgD and IgM expressions. Left panels (WILD/F759)show the results of control mouse (gp130^(WT/F759) mouse) and rightpanels (F759/F759) show the results of gp130^(F759/F759) mouse. Thenumbers in the panels indicate the frequency (%) of the cell populationwithin areas boxed or separated by cross lines.

FIG. 8 shows the results of flow cytometry analysis of a lymph node ofgp130^(F759/F759) mouse. Lymph node cells of a mouse were multiplystained with fluorescence-labeled monoclonal antibodies, and thefluorescence intensities were measured by flow cytometer. Vertical andlateral axes indicate the fluorescence intensities reflecting thebinding of the fluorescence-labeled antibodies against the surfaceantigens, which are indicated in the vertical and lateral direction onthe left side of the panels, respectively, and thus, the expressionlevels of the surface antigens are indicated for the individual cells.Upper panels: the results of analysis of CD4 and CD8 expressions. Middlepanels: the results of analysis of IgD and IgM expressions. Lowerpanels: the results of analysis of Gr-1 and CD11 expressions. Leftpanels (WILD/F759) show the results of control mouse (gp130^(WT/F759)mouse) and right panels (F759/F759) show the results ofgp130^(F759/F759) mouse. The numbers in the panels indicate thefrequency (%) of the cell population within areas boxed or separated bycross lines.

FIG. 9 shows the results of flow cytometry analysis of CD4 and CD8single positive cells in a lymph node of gp130^(F759/F759) mouse. CD4 orCD8 single positive cells in a lymph node of a mouse were multiplystained with a fluorescence-labeled monoclonal antibodies, and thefluorescence intensities were measured by flow cytometer. Vertical andlateral axes indicate the fluorescence intensities reflecting thebinding of the fluorescence-labeled antibodies against the surfaceantigens, which are indicated in the vertical and lateral direction onthe left side of the panels, respectively, and thus, the expressionlevels of the surface antigens are indicated for the individual cells.Upper panels: the results of analysis of CD69 and CD25 expressions inCD4 single positive cells. Middle panels: the results of analysis ofCD62L and CD44 expressions in CD4 single positive cells. Lower panels:the results of analysis of CD62L and CD44 expressions in CD8 singlepositive cells. Left panels (WILD/F759) show the results of controlmouse (gp130^(WT/F759) mouse) and right panels (F759/F759) show theresults of gp130^(F759/F759) mouse. The numbers in the panels indicatethe frequency (%) of the cell population within areas boxed or separatedby cross lines.

BEST MODE FOR CARRYING OUT THE INVENTION

The term “tyrosine residue corresponding to the tyrosine residue atposition 759 of human gp130 protein” as used herein means a tyrosineresidue in gp130 protein of the transgenic mouse of the invention,wherein said tyrosine residue corresponds to the tyrosine residuelocated at position 759 in human gp130 protein. The term “regioncomprising a tyrosine residue corresponding to the tyrosine residue atposition 759 of human gp130 protein” means a region of the amino acidresidues in the gp130 protein of the transgenic mouse of the invention,wherein said region comprises said tyrosine residue corresponding to thetyrosine residue at position 759 of human gp130 protein. In a gp130variant expressed in the transgenic mouse of the invention, a tyrosineresidue, which corresponds to the tyrosine residue at position 759 ofhuman gp130 protein, is substituted or deleted, and/or one or more aminoacid residue are substituted, inserted or deleted in a region comprisingsaid corresponding tyrosine residue.

gp130 variant expressed in the transgenic mouse which is used in themethod of screening of the invention may also have any mutation so thatthe functions of any other unknown signaling molecule, which requires atyrosine residue corresponding to the tyrosine at position 759 of humangp130 protein as well as the bindings of SHP2 and SOCS-3, can beinhibited. Thus, in the transgenic mouse used in the screening method ofthe invention, the SHP2-mediated gp130-dependent signaling cascade isselectively disrupted, the feedback control of cytokine signaling bybinding of SOCS-3 does not work, and the functions of any unknownsignaling molecule requiring a tyrosine residue, which corresponds tothe tyrosine residue at position 759 of human gp130 protein, areinhibited.

In the present invention, gp130 variant expressed in the transgenicmouse of the invention comprises substitution, insertion, or deletion ofone or more amino acid residues in a region comprising a tyrosineresidue, which corresponds to the tyrosine residue at position 759 ofhuman gp130 protein. The tyrosine residue itself, which corresponds tothe tyrosine residue at position 759 of human gp130 protein, may be ornot be substituted, so long as the functions of the signaling moleculerequiring said tyrosine residue are inhibited.

In a preferred embodiment of the invention, gp130 variant expressed inthe transgenic mouse of the invention comprises substitution or deletionof a tyrosine residue, which corresponds to the tyrosine residue atposition 759 of human gp130 protein.

In a more preferred embodiment of the invention, gp130 variant expressedin the transgenic mouse of the invention comprises substitution of atyrosine residue, which corresponds to the tyrosine residue at position759 of human gp130 protein, by a point mutation of the gp130 gene.

The term “point mutation” refers to a mutation whereby a single basepair on a DNA molecule is substituted with any different base pair. Thepoint mutation may be directly introduced into the gp130 gene in mousegenome, or introduced by homologous recombination between a fragment ofmutant gp130 gene containing such point mutation and the gp130 gene ofmouse genome. The point mutation specifically preferable in the methodof the invention is, for example, such point mutation as phenylalanineis encoded. As used herein, the transgenic mouse wherein a mutant gp130gene having such point mutation is introduced is referred to as“gp130^(F759) mouse”, and a homozygote of the mutant gp130 gene isespecially referred to as “gp130^(F759/F759) mouse”. Further, aheterozygote between the wild-type gp130 gene and the mutant gp130 geneis referred to as “gp130^(WT/F759) mouse”, and a homozygote of thewild-type gp130 gene is referred to as “gp130^(WT/WT) mouse”.

The transgenic mouse used in the screening method of the invention maybe prepared in the following manner. A mutant gp130 gene is prepared bydirect introduction of the above mutation into the gp130 gene of mouseor by homologous recombination between the fragment of mutant gp130 geneas described above and the gp130 gene of mouse genome, and the mutantgp130 gene obtained is then introduced into a mouse ES cell usingprocedures well known in the art to establish an ES cell comprisingmutations on the gp130 genome. Then, the ES cells is introduced into ablastocyst cell taken from another mouse, the blastocyst cell isintroduced into a uterus of a pseudopregnant mouse, and a chimeric mouseborn of the pseudopregnant mouse and a wild-type mouse are crossed toproduce the first generation of heterozygous mice. The heterozygous miceof the first generation are then crossed with each other to produce thesecond generation of homozygous mice. The transgenic mouse used in thescreening method of the invention may be prepared, for example,according to the procedures described in Immunity, Vol. 12, 95–105,January, 2000.

A test compound which may be used in the screening method of theinvention includes, for example, peptides, proteins, non-peptidecompounds, antisense DNAs, antisense RNAs, synthetic compounds,fermented products, cell extracts, plant extracts, mammal tissueextracts, plasma, serum and the like, and these test compounds may benovel or known.

The means for administration of a test compound in the screening methodof the invention includes, for example, oral administration, intravenousinjection, intradermal injection, intramuscular injection and the like.Those skilled in the art can appropriately select the dosage of a testcompound according to the route of administration, the property of thetest compound and the like. The administration of a test compound may bestarted prior to the timing of the onset of immune abnormality or thedisease for screening of prophylactic drugs, whereas after the diseaseis macroscopically presented for screening of therapeutic agents.

In the screening method of the invention, male and female wild and thetransgenic mice of the invention are divided into groups, and theanimals, with or without administration of a test compound, are visuallyobserved and scored for the severity of the symptom. Blood was collectedfrom the mice and subjected to blood cell counting and serological atest, additionally a urine test and the like are also conducted. After acertain period of observation, X-ray photogram analysis, pathologicalanalysis and immunological analysis are conducted. If any mouse diedfrom unknown cause during the test period, pathological autopsy isconducted to clarify the cause.

Tissues or cells are isolated from the transgenic mouse of the inventionand subjected to analyses in vitro to assess preventing or therapeuticeffect of a test compound, changes in the cellular phenotype resultingfrom the genetic modification for changes in cell growth and cytokineproduction and the like.

Further, the transgenic mouse of the invention may be crossed to producevarious mice of different genetic backgrounds, so that it is possible toassess influences in development of the diseases and to try variousinduced model mice to establish experimental systems permitting morerapid screenings for preventing agents or therapeutic agents.

The examples described below should be construed to illustrate thepresent invention, and not to limit the scope of the invention in anyway.

EXAMPLES

Preparation of gp130^(F759/F759) Mouse

In accordance with the method as described in Immunity, Vol. 12, 95–105,January, 2000, the EcoRI-XhoI fragment of human gp130 cDNA containingthe point mutation that is a substitution of tyrosine 759 withphenylalanine (mutation from TAT to TTT) (Yamanaka, Y et al. (1996) EMBOJ. 15, 1557–65), wherein the XhoI site was created by the subcloning,was introduced into mouse gp130 gene to provide mouse-human chimericgp130 constructs. The targeting vectors bearing the constructs weretransduced into mouse ES cells by electroporation, and the ES cellscontaining the mutation in gp130 genome were established. Using themicroinjection method, the transgenic ES cells were injected intoblastocysts that had been taken from C57BL/6 mice. The blastocystsinjected with the ES cells were transplanted into a uterus of apseudopregnant mouse (ICR line) as obtained by intercoursing with amouse subjected to vasoligation. Chimeric mice of the newborns from thepseudopregnant mouse were crossed with C57BL/6 wild-type mice to providethe first generation hetero mice. Crossing between the first generationhetero mice provided the second generation homozygous mice(gp130^(F759/F759) mice).

Analysis for Pathophysiology of gp130^(F759/F759) Mouse

Production of autoantibodies:

Blood was collected from gp130^(F759/F759) mice at the age of 5, 9 and12 months, and the serums were separated. Production levels ofautoantibodies such as immunoglobulin, anti-ssDNA (single-strandedDNA)-, anti-dsDNA (double-stranded DNA)-, anti-nRNP(ribonucleoprotein)-antibodies, and rheumatoid factors were determinedby ELISA method. The productions of autoantibodies such as anti-ssDNA-and anti-dsDNA-antibodies were found in the mice aged 9 months and more.The results of the determinations in the mice at the age of 5, 9 and 12months are shown in FIG. 1.

Development of arthritis:

gp130^(F759/F759) mice were observed for the development of arthritisuntil 20 months after birth. Twenty % of the gp130^(F759/F759) mice aged8–10 months developed arthritis, whereas the gp130^(F759/F759) mice aged20 months developed arthritis at near 100% (FIG. 2). FIG. 3 shows themacroscopic observation of the arthritis in the gp130^(F759/F759) mice(aged 9.5 months). In the gp130^(F759/F759) mice, the swelling and therestricting range of motion in the proximal finger joints of theextremities were found, and the arthritis attacked even the majorjoints, then causing the ankylosing changes. After the gp130^(F759/F759)mice that developed arthritis were sacrificed, the joints, the thymusgland, the spleen, the liver or the lymph node was removed, and fixed onformalin. Then, the pathological samples were prepared according toconventional manners, stained with hematoxylin and eosin, and subjectedto the pathological examination with an optical microscope. FIGS. 4–6show the results of the histopathological examination of thegp130^(F759/F759) mice. The gp130^(F759/F759) mouse were outstanding infibrin deposition, neutrophilic infiltration, and joint structuredestruction associated with fibroblasts growth in the synovial membrane(FIGS. 4 and 5). Further, plasma cell infiltration around the Glisson'scapsule of the liver (9.5-month-old: the right upper in FIG. 6), and thehepatic cell degeneration (15.5-month-old: the right medium in FIG. 6)were observed. Also, plasma cell infiltration in the lymph node wasobserved (the right lower in FIG. 6).

Aberrations in lymphatic tissues (analysis by flow cytometry):

Cells were removed from the lymphatic tissues (thymic gland and lymphnode), and were examined for the cell surface markers and antibodies toeach specific surface antigens with a cell surface analyzer. In thethymic gland, T cells expressing both CD4 and CD8 (CD4/CD8double-positive T cells) were decreased (2% in the gp130^(F759/F759)mice, whereas 83% in the control: the upper in FIG. 7), and T cellsexpressing singly either CD4 or CD8 (CD4 single-positive T cells, CD8single-positive T cells) were increased (31% and 14% in thegp130^(F759/F759) mice, respectively, whereas 8% and 4% in the control:the upper in FIG. 7). Further, unusual proliferation of B cell(IgD+IgM^(lo) (lower expression of IgM)) was observed in the thymicgland of the gp130^(F759/F759) mice (32% increase: the lower in FIG. 7),which was not found in normal thymic gland. In the lymph node of thegp130^(F759/F759) mice, Gr-1+CD11b+ granulocytes were increased (6%: thelower in FIG. 8). Additional examination on the CD4 single-positive Tcells in the lymph node of the gp130^(F759/F759) mice revealed that thecells positive at marker CD69 or CD25 indicate that activated cells wereincreased (the upper in FIG. 9). Examination on the CD62L and CD44expressions revealed that there was a decrease in naive T cells(CD62L+CD44−) (3% in the gp130^(F759/F759) mice, whereas 58% in thecontrol), and the increase in activated memory T cells (CD62L−CD44+)(71% in the gp130^(F759/F759) mice, whereas 25% in the control) wereobserved among the CD4 single-positive T cells in the lymph node of thegp130^(F759/F759) mice (the medium in FIG. 9). Among the CD8single-positive cells, the decrease in naive T cells (CD62L+CD44−) 2% inthe gp130^(F759/F759) mice, whereas 49% in the control: the lower inFIG. 9) was observed. These results show that the T cells in the lymphnode were unusually activated in the gp130^(F759/F759) mice, whichdeveloped the arthritis.

Test Example 1

Screening Test for Anti-Arthritis Agents

gp130^(F759/F759) mice or mice obtained by backcrossing of thegp130^(F759/F759) mice (gp130^(F759/F759)-related mice) were used as amodel mouse. A test compound of antirheumatic drugs such as bucilamineand methotrexate, nonsteroidal anti-inflammatory drugs such asdiclofenac, or steroidal drugs such as dexamethasone was administered.The administration was conducted before the onset of immune disorders orarthritis for screening of prophylactic drugs, whereas after theobservation of visible arthritis for screening of therapeutic drugs. Thefollowing is to illustrate the screening test for anti-arthritic agentsusing methotrexate as a test compound, and screening tests for othercompounds would be conducted according to a similar manner to thefollowing.

Methotrexate was administered orally at a dose of 0.4 mg/kg to the miceat the aged of 9 months for five days in a week. Male or female wild orgp130^(F759/F759) mice were divided into groups of 2 or 3 animals pergroup, which were observed with naked eye for the course of theprophylactic or therapeutic effect on the arthritis. The severity ofarthritis was scored based on restricted range of motion, swelling andflare of digit joints, and the presence or absence of rigidity. The micewhose arthritis conditions were observed constantly for two weeks ormore were estimated as developed animal, and incident and severity weredetermined with time course.

Consequently, in the control (without methotrexate), the animals at theage of 40 weeks or older (about 10-month-old) were found to have thearthritis, and all of the animals aged 46 weeks developed the arthritis.Further estimation of the arthritis score in the mice developingarthritis with time course revealed that the animals aged 48 weeks(12-month-old) and 53 weeks in the group receiving methotrexate (0.4mg/kg) showed 1.33±0.33 and 0.67±0.33, respectively, whereas the animalsaged 48 weeks (12-month-old) and 53 weeks in the control showed4.33±1.86 and 4.67±0.67, showing that methotrexate inhibits thearthritis in the mouse model of the present invention.

Test Example 2

Screening Test for Anti-Inflammatory Drugs

Using gp130^(F759/F759) mice or their related mice, the air pouch modelis prepared to screen for a compound having an inflammatory activity.Candidate compounds possibly having an inflammatory activity areadministered to the air pouch model as prepared according to aconventional manner before or after inducing inflammation, and theinfiltrations of neutrophil and macrophage in the air pouch areexamined. Further, the changes in phenotypes in gp130^(F759/F759) micecaused by other inflammatory-inducing agents are examined in a similarmanner to the method of Test Example 1.

Test Example 3

Screening Test for Antiinfection Drugs

gp130^(F759/F759) mice are so sensitive to listeriosis that they areoften die due to the infection. Accordingly, gp130^(F759/F759) mice canbe used to screen for a compound having a therapeutic activity forinfections. For example, the mice are infected with Listeria, a testcompound is administered thereto, and the therapeutic effect is examinedusing survival rate as an index.

Test Example 4

Screening Test for Drugs Controlling an IL-6-Family Cytokine Signal.

It is believed that gp130^(F759/F759) mice would develop diseases causedby overexpression of STAT3 that involves signal transmission mediated byIL-6. Accordingly, gp130^(F759/F759) mice can be used to screen for acompound that prevent or treat diseases caused by aberration in signaltransmission mediated by IL-6. The tissues, the cells, the genes or theproteins derived from the mice are also used to screen for various drugsthat control the signal.

The invention claimed is:
 1. A method of screening for substances fortreating arthritis in association with IL-6 family cytokine receptormalfunction, said method comprises using a transgenic mouse expressing agp130 variant, wherein said gp130 variant comprises a substitution,which corresponds to the tyrosine residue at position 759 of human gp130protein, in which the tyrosine is substituted by a phenylalanine.
 2. Amethod of screening for substances for treating rheumatoid arthritis inassociation with IL-6 family cytokine receptor malfunction, said methodcomprises using a transgenic mouse expressing a gp130 variant, whereinsaid gp130 variant comprises a substitution, which corresponds to thetyrosine residue at position 759 of human gp130 protein, in which thetyrosine is substituted by a phenylalanine.